ABSTRACT

The interconversion of oestrone and oestradiol, androstenedione and testosterone, and dehydroepi-androsterone and 5-androstene-3β,17β-diol in mammalian tissues is catalysed by 17β-hydroxysteroid oxidoreductase (17β-HSOR). To identify tissue sites of 17β-HSOR activity in the human fetus, microsomal fractions from 15 different fetal tissues obtained from first and second trimester pregnancies were used for evaluation of enzymatic activity by use of [17α-³H] oestradiol as the substrate and NADP⁺ as the co-factor. With these reagents, the enzyme-catalysed reaction led to the production of both non-radiolabelled oestrone and NADP³H in equimolar amounts; the radioactivity associated with NADP³H was used to quantify 17β-HSOR activity. Activity of 17β-HSOR was present in microsomes of all the tissues evaluated. The specific activity of the enzyme was highest in liver and placental microsomes. The interconversion of oestradiol and oestrone in microsomal fractions of nine different fetal tissues was studied by the use of substrates labelled with tritium at stable nuclear positions ([6,7-³H]oestradiol and [6,7-³H]oestrone). The products, [³H]oestrone and [³H]oestradiol, were quantified by the use of established techniques; other metabolites formed in these incubations were not identified. The reductive pathway of metabolism (oestrone to oestradiol) appeared to be favoured in microsomal fractions prepared from placenta, fetal zone of the adrenal gland and, possibly, lung. The oxidative pathway (oestradiol to oestrone) appeared to be favoured in microsomes prepared from liver, intestine, stomach, kidney, brain and heart. 17β-HSOR activity in fetal liver also was assessed by the use of fresh and frozen-thawed tissue, homogenate, subcellular fractions, and, also, in primary hepatocytes maintained in culture; the specific activity of the enzyme was highest in the microsomal fraction of liver tissue and 17β-HSOR activity in liver microsomes was linear with time of incubation up to 1 h. In hepatocytes, the enzymatic activity was linear with time of incubation up to 2 h and with cell number up to 2·⁵ × 105 cells/ml; the apparent Michaelis constant of hepatocyte 17β-HSOR for oestradiol was 11 μmol/l. The specific activity of 17β-HSOR did not change after pretreatment of hepatocytes for 24 h with insulin, glucagon or dexamethasone.

Journal of Endocrinology (1989) 123, 509–518

Corticosteroid control of distal nephron sodium handling, particularly through the amiloride-sensitive sodium channel (ENaC), has a key role in blood pressure regulation. The mechanisms regulating ENaC activity remain unclear. Despite the generation of useful mouse models of disorders of electrolyte balance and blood pressure, there has been little study of distal nephron sodium handling in this species. To investigate how corticosteroids regulate ENaC activity we isolated cDNA for the three mouse ENaC subunits (alpha, beta and gamma), enabling their quantitation by competitive PCR and in situ hybridisation. Kidneys were analysed from mice 6 days after adrenalectomy or placement of osmotic mini-pumps delivering aldosterone (50 microg/kg per day), dexamethasone (100 microg/kg per day), spironolactone (20 mg/kg per day) or vehicle alone (controls). In controls, renal ENaCalpha mRNA exceeded beta or gamma by approximately 1.75- to 2.8-fold. All subunit mRNAs were expressed in renal cortex and outer medulla, where the pattern of expression was fully consistent with localisation in collecting duct, whereas the distribution in cortex suggested expression extended beyond the collecting duct into adjacent distal tubule. Subunit mRNA expression decreased from cortex to outer medulla, with a gradual reduction in beta and gamma, and ENaCalpha decreased sharply ( approximately 50%) across the outer medulla. Expression of ENaCbeta and gamma (but not alpha) extended into inner medulla, suggesting the potential for inner medulla collecting duct cation channels in which at least ENaCbetagamma participate. Aldosterone significantly increased ENaC subunit expression; the other treatments had little effect. Aldosterone caused a 1.9- to 3.5-fold increase in ENaCalpha (particularly marked in outer medullary collecting duct), but changes for beta and gamma were minor and limited to the cortex. The results raise the possibility that medullary ENaCalpha upregulation by aldosterone will create more favourable subunit stoichiometry leading to a more substantial increase in ENaC activity. In cortex, such a mechanism is unlikely to have a major role.

Abstract

A tumour of the left adrenal gland was identified in a woman who presented with virilization and secondary amenorrhea. Preoperatively, the plasma levels of dehydroepiandrosterone sulphate, dehydroepiandrosterone, androstenedione, testosterone, 5α-dihydrotestosterone and 5-androstene-3β,17β-diol were elevated two- to fourfold whereas those of urinary 17-ketosteroids were elevated more than tenfold. The production rate of dehydroepiandrosterone sulphate was more than 16 times that in normal women whereas those of dehydroepiandrosterone, testosterone and androstenedione were approximately twofold greater; plasma testosterone was derived almost entirely from the peripheral conversion of androstenedione. Blood was obtained by catheterization of the ovarian veins, left adrenal gland vein and inferior vena cava (at two different sites) and plasma steroid levels were determined: testosterone and cortisol levels were elevated in all blood samples whereas those of androstenedione, dehydroepiandrosterone sulphate and 11-desoxycortisol were approximately six- to eightfold, 1·5-fold and nine- to 22-fold higher in the effluent of the left adrenal gland/tumour compared with the levels in the other compartments. Blood was collected hourly for 24 h to determine steroid levels under basal conditions and, also, after ACTH treatment. Plasma cortisol levels increased markedly upon ACTH administration and fell to very low levels 11 h later, but those of androstenedione, testosterone, dehydroepiandrosterone, 5-androstene-3β,17β-diol and dehydroepiandrosterone sulphate were not affected by ACTH treatment. A histological diagnosis of cortical adenoma of the extirpated tumour was made. Tissue explants and adenoma cells were maintained in culture to characterize the steroid-metabolizing properties of the tumour. The secretion of dehydroepiandrosterone sulphate by tissue explants was high initially, but declined to almost undetectable levels after 5 days in culture. In the presence of ACTH, dehydroepiandrosterone sulphate secretion remained elevated throughout the entire study up to 5 days. Basal secretion of dehydroepiandrosterone sulphate, androstenedione, 11-desoxycortisol, cortisol, testosterone and 11β-hydroxyandrostenedione by adenoma cells was either very low or undetectable. In the presence of ACTH, dibutyryl cyclic AMP or cholera toxin the secretion of dehydroepiandrosterone sulphate, androstenedione and 11-desoxycortisol increased markedly with time in culture up to 3 days, whereas the other steroids were undetected in the medium. A homogenate of adenoma tissue metabolized testosterone to androstenedione, but the conversion of androstenedione to testosterone was minimal. The findings of this study served to establish that virilization in this woman was due, at least in part, to excess testosterone – and testosterone-derived 5α-dihydrotestosterone – produced at extra-adrenal tissue sites almost exclusively through metabolism of tumour-secreted androstenedione. The excess production of steroid prohormones in this woman was due to autonomous tumour steroidogenesis. The remarkable feature was the degree of virilization resulting from a modest increase in biologically potent androgens.

Journal of Endocrinology (1994) 140, 297–307